Gram-positive pathogens such as Staphylococcus aureus, Streptococcus pyogenes and Enterococcus faecalis are responsible for a number of deadly diseases. The ubiquitous S. aureus is a leading cause of hospital acquired infections (over 700,000 annually); S. pyogenes is the organism responsible for severe Group A streptococcal infection which has mortality rates in excess of 60%; and E. faecalis has become an important health concern as a mediator of the spread of antibiotic resistance and a leading cause of nosocomial infections. During the last decade widespread emergence of antimicrobial resistance in these organisms has been recognized as one of the major health threats for mankind. New antimicrobial agents are urgently needed to advert a coming plague of multi-drug resistant gram-positive pathogens. [unreadable] [unreadable] The long term objective of our work in this area is to provide a structural foundation for understanding how gram positive organisms cause disease. Such an understanding can lead to the development of new pharmaceutical agents and vaccines. The pathogenicity of staphylococci, streptococci and enterococci is a result of a remarkable array of virulence mechanisms by which they attack their hosts. These mediate: (1) adherence to host cells and tissue, (2) evasion of host defenses, (3) invasion of host tissue, and (4) dissemination of antibiotic resistance. Previously, we determined the structures of a number of toxin superantigenic virulence factors, including toxic shock syndrome toxin-1 and exfoliative toxins A and B from S. aureus and streptococcal pyrogenic exotoxin A from S. pyogenes. Here we propose to determine the structures of five virulence factors and relevant physiological complexes representative of the four key virulence mechanisms: PrgX, a pheromone response regulator; PrgB, an aggregation substance protein found in pheromone-mediated conjugative plasmids, streptococcal C5a peptidase which targets the chemotactic anaphylatoxin C5a; a SspC, a protease inhibitor specific for a major staphylococcal secreted cysteinyl protease, SspB; and, bhemolysin, a sphingomyelinase. Of the 5 targets chosen, 4 have already yielded diffraction-quality crystals. [unreadable] [unreadable] [unreadable]